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Adenoassociated virus vectors for the treatment of lysosomal storage disorders

a technology of lysosomal storage and virus vector, which is applied in the direction of drug composition, peptide/protein ingredient, metabolic disorder, etc., can solve the problems of progressive intellectual decline, severe behavioral problems, and particularly afflicted groups in the general population

Active Publication Date: 2017-03-30
AUTONOMOUS UNIVERSITY OF BARCELONA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides new methods for treating diseases, specifically mucopolysaccharidoses type III (MPSIII). The invention involves the use of adenoassociated virus vectors (AAV) containing a nucleotide sequence coding for N-acetylglucosaminidase, alpha (Naglu). The AAV vectors have been shown to be efficient in reversing the storage of pathological GAGs in brains and somatic tissues. The invention also includes plasmid vectors containing the same nucleotide sequence, as well as pharmaceutical compositions containing these vectors or plasmid vectors. The invention also includes methods for delivering the vectors and the isolated cells used in the invention. Overall, the invention provides effective treatments for MPSIII and offers a promising approach for the development of new therapies for rare diseases.

Problems solved by technology

However, some groups within the general population are particularly afflicted by a high occurrence of LSDs.
This is followed by severe behavioral problems and progressive intellectual decline during the second phase of the disease.
Finally, with the onset of severe dementia, the behavioral problems slowly disappear, and all motor functions start to decline, eventually resulting in dysphagia, complete loss of locomotion and pyramidal tract lesions.
On the other hand, melatonin administration is considered to be the best treatment for the sleep disorders characteristic of the disease, although it is not completely effective.
However, subsequent studies did not show improvement in either the disability scales or the behaviour scores of MPSIIIA, MPSIIIB or MPSIIIC patients treated with genistein for 12 months.
However, the implantation of the permanent intrathecal delivery device that the therapy requires is associated with substantial risks and shortcomings, and the therapy itself has a very high economic cost per patient / year.
However, no clear benefit has been observed in Sanfilippo A or B patients who have undergone BMT.
Therefore, HSCT using BMT is currently not considered as a treatment option for MPSIII patients.
Lentiviral vectors coding for the human N-acetylglucosaminidase, alpha gene have been administered intravenously to young MPSIIIB mice, resulting in low levels of transgene expression in liver, spleen, lung and heart, which reduced but did not normalize GAG accumulation in these tissues.
These vectors have, however, a low distribution within the brain parenchyma, and the approach requires multiple injections.
However, enzymatic activity remained low or undetectable in the most rostral and most caudal regions, especially in the cerebellum.
Lysosomal pathology was improved but not fully corrected in treated MPSIIIB dogs, indicating that the levels of enzymatic activity achieved with this approach were insufficient to cope with GAG storage.
The larger the brain the more difficult it becomes to cover the whole volume of the organ with intraparenchymal injections, and delivery to humans needs vector administration at several sites, making delivery technically challenging and requiring the development of specific surgical procedures.
The use of such high doses would suppose a challenge for clinical translation from the manufacturing and safety points of view.

Method used

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  • Adenoassociated virus vectors for the treatment of lysosomal storage disorders
  • Adenoassociated virus vectors for the treatment of lysosomal storage disorders
  • Adenoassociated virus vectors for the treatment of lysosomal storage disorders

Examples

Experimental program
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Effect test

example 1

ion of pAAV-CAG-hNaglu

[0145]The human N-acetylglucosaminidase, alpha coding sequence (CDS) was utilized as starting material (NCBI Reference Sequence: NM_000263) and chemically synthesized for this purpose (GeneArt; Life Technologies). The CDS was received cloned inside the plasmid pMA (AmpR) flanked by MluI and EcoRI restriction sites at 5′ and 3′ ends, respectively. N-acetylglucosaminidase, alpha CDS was excised by MluI / EcoRI digestion and then cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG (AmpR). The resulting plasmid was named pAAV-CAG-hNaglu (accession number DSM 28568). See SEQ ID NO: 5, and FIG. 19 A.

[0146]The pAAV-CAG plasmid had been previously generated and contained the ITRs from the AAV2 genome, the CAG promoter, and the polyA signal from rabbit β-globin, as well as a multicloning site for cloning of CDSs of interest. The CAG promoter is a hybrid promoter composed of the CMV early / intermediate enhancer and the chicken β-actin p...

example 2

n of AAV9-CAG-hNaglu

[0147]Vectors AAV9-CAG-hNaglu (SEQ ID NO: 9 and FIG. 19 B)) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra. Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, N.Y., US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-hNaglu); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions. Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at −80° C. until use.

example 3

ion of pAAV-CAG-cohNaglu

[0148]Expression cassettes including an optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu) were designed and obtained. The sequence optimization (GeneArt®) was performed to maximize the efficiency of N-acetylglucosaminidase, alpha protein production in human beings through elimination of cryptic splice sites and RNA destabilizing sequence elements for increased RNA stability, addition of RNA stabilizing sequence elements, codon optimization and G / C content adaptation, avoidance of stable RNA secondary structures amongst others changes. The optimized CDS was received cloned in the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5′ and 3′, respectively.

[0149]The pMA-RQ-cohNaglu plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu...

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Abstract

The present invention provides new adenoassociated virus vectors and pharmaceutical compositions containing the same for the treatment of lysosomal storage disorders and specially, for the treatment of mucopolysaccharidoses Type IIIB.

Description

FIELD OF THE INVENTION[0001]The present invention relates to vectors useful for the expression of proteins of interest and their utilization in gene therapy. The present invention also relates to vectors and nucleic acid sequences helpful for the treatment of mucopolysaccharidoses (MPS), and in particular, for the treatment of mucopolysaccharidoses type III-B or Sanfilippo B syndrome.BACKGROUND OF THE INVENTION[0002]The lysosome is an organelle found in the cytoplasm of animal cells that contains more than 50 hydrolases that break down biomolecules during the recycling of worn-out cellular components or after the engulfment of viruses and bacteria. This organelle contains several types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All enzymes are acid hydrolases.[0003]Lysosomal storage diseases (LSDs) are caused by genetic defects that affect one or more lysosomal enzymes. These genetic diseases result gene...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86A61K38/47C12N9/26
CPCC12N15/86C12N9/2474C12Y302/0105A61K48/00C12N2750/14143C12N2830/008C12N2840/105A61K38/47A61K9/0019A61K9/0085A61K48/005C12N9/2402A61K48/0075C12N2800/22A61P1/16A61P13/00A61P19/08A61P25/00A61P3/00A61P43/00
Inventor BOSCH TUBERT, M FATIMAHAURIGOT MENDO A, M VIRGINIARIBERA SANCHEZ, ALBERT
Owner AUTONOMOUS UNIVERSITY OF BARCELONA
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